Method for simultaneously determining human cyclosporine action target gene polymorphism

A gene polymorphism and cyclosporine technology, applied in the field of molecular biology, can solve the problems of unsuitable detection, high cost, time-consuming and labor-intensive, etc., and achieve the effects of simple operation, high sensitivity and low analysis cost.

Inactive Publication Date: 2015-09-23
AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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Problems solved by technology

The direct DNA sequencing method is the gold standard for mutation detection, but it is time-consuming, labor-intensive, and expensive. It is a low-throughput detection method and is not suitable for detection of a large number of samples.
The Taqman is a highly specific quantitative PCR technology, which is characterized by being suitable for the detection of a small number of SNP sites in large samples, and is a medium-throughput SNP detection. However, for simultaneous detection of multiple SNP sites, the cost is relatively high, and it is not necessary suitable

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  • Method for simultaneously determining human cyclosporine action target gene polymorphism
  • Method for simultaneously determining human cyclosporine action target gene polymorphism
  • Method for simultaneously determining human cyclosporine action target gene polymorphism

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Experimental program
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Embodiment 1

[0080] The genotypes of 5 genes including PPIA, PPP3CB, PPP3R1, NFATC1, and NFATC2 totaling 78 SNP loci were detected by time-of-flight mass spectrometry in 10 renal transplant patients.

[0081] 1. Genomic DNA extraction from human whole blood

[0082] Extract genomic DNA from whole blood with a DNA extraction kit, and adjust the concentration to 10-100ng / ul.

[0083] 2. Multiplex PCR reaction

[0084] 1) Find the target gene sequence, design and synthesize PCR primers for the mutation site

[0085] Simultaneous determination of 78 SNP sites in PPIA, PPP3CB, PPP3R1, NFATC1, and NFATC2 genes, and the designed primers are shown in Table 1.

[0086] 2) Prepare a 384-well reaction table based on the extracted samples, and indicate the number of the DNA sample corresponding to each well and the primers used.

[0087] 3) According to the table, add 1 μL of DNA template to each well of the 384-well plate, stick to the film, and centrifuge at 2000 rpm / 10 seconds for later use.

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Abstract

The invention provides a method for simultaneously determining human cyclosporine action target gene polymorphism, namely, a method for simultaneously determining cyclosporine action target pathway series protein coding gene multiple single nucleotide polymorphism (SNP) locus genotypes. Operation is simple and convenient, sequence specific probes are not required, multiple detection of 384 samples can be carried out by one chip, 40 reactions can be realized by each system at most, meanwhile, 40 SNP loci can be detected simultaneously, the flux can be adjusted individually according to requirements, the high flux is realized, the application range is wide, dozens of samples to tens of thousands of samples can be detected, and dozens of loci to hundreds of loci can be detected simultaneously, fluorescence labeling is not required, only common primers are required to be synthesized and the individual analysis cost is low; high flexibility is realized since samples and locus detection matching on one chip can be selected randomly; the sample size required for analysis (10 ng) is small and high sensitivity and high detection accuracy are realized.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a simultaneous determination of multiple single nucleotide polymorphism (single nucleotide polymorphism, SNP) site genes in the protein coding genes of the cyclosporine (Cyclosporine, CsA) action target pathway series A type of method, that is, a method for simultaneously determining the polymorphism of the target gene of human cyclosporine. Background technique [0002] The therapeutic window of cyclosporine is narrow, and there are significant individual differences in the curative effect. At present, the dose of cyclosporine is usually adjusted clinically through therapeutic drug monitoring (therapeutic drug monitoring, TDM), that is, monitoring blood drug concentration. However, traditional TDM is difficult to reflect the variation among individuals from the perspective of pharmacodynamics, and TDM can only be implemented after taking the medicine, and it is impossi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888C12Q2600/106C12Q2600/156
Inventor 邱晓燕徐勤霞焦正钟明康
Owner AFFILIATED HUSN HOSPITAL OF FUDAN UNIV
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